A considerable body of evidence has accumulated in recent years implicating the beta-amyloid protein (Abeta) in the etiology of Alzheimer's disease (AD). Abeta is a proteolytic fragment derived from an integral membrane protein called the amyloid precursor protein (APP). In particular, a specific Abeta variant is thought to be an essential element in AD pathogenesis: while approximately 90 percent of Abeta secreted from cells is the 40-amino acid C-terminal variant (Abeta40), the major protein constituent in neuritic AD plaques is the 42-amino acid C-terminal variant (Abeta42). This longer Abeta is much more susceptible to nucleation and fibril formation, and all mutations associated with familial early-onset AD (FAD) examined to date have been shown to cause increases in Abeta42. Little is known about the enzymes responsible for cleaving Abeta from APP. Recent evidence suggests that different protease activities are associated with the formation of Abeta40 and Abeta42 and these activities have been dubbed gamma(40)-secretase and gamma(42)-secretase, respectively. Through inhibitor design, it is the aim of this project to decipher the determinants of the APP/gamma-secretase interactions and to create molecular tools for isolating the gamma-secretases and understanding their role in normal and pathological cellular events. In addition, such inhibitors may help directly address the amyloid hypothesis of AD and serve as prototypes for drug development. The P.I. has identified certain peptidomimetic transition-state analogs, designed based on the APP amino acid sequence, as specific inhibitors of gamma-secretases. Questions to be addressed in the proposed work include: (1) How can these peptide analogs be modified to attain selective and potent inhibition of Abeta42? (2) How can these peptide analogs be modified to attain selective and potent inhibition of Abeta40? (3) Do the analogs suggest how APP and gamma-secretases interact? (4) Do the analogs suggest in what class of proteases the gamma-secretase belong? (5) Do the analogs provide information that help elucidate the biochemical mechanisms by which Abeta is formed?